The H+:lactose carrier of Escherichia coli is a representative of a broad class of membrane proteins known as cation-substrate cotransporters. The primary aim of the proposed research is an understanding of the relationship between the protein structure of the H+:lactose carrier and its physiological function of transporting H+ and lactose across the membrane. Lactose carrier mutants will be isolated with alterations in their sugar recognition site. These "sugar-specificity" mutants will be subjected to DNA sequencing in order to identify amino acid residues which are close to, or a part of, the sugar recognition site. Amino acids which are postulated to be close to the sugar recognition site will be modified by group-specific reagents to determine their proximity to that site and their role during sugar recognition and transport. With regard to the H+ recognition site, negatively charged amino acid residues will be replaced with noncharged residues via site-specific mutagenesis. The "site- directed" mutants will be tested for their ability to transport H+ in order to identify negatively charged amino acids which are directly involved with H+ recognition and transport. Finally, to elucidate details about the structure of the protein, regions of the lactose carrier which project into the cytoplasm will be localized by isolating and sequencing lactose carrier mutants which are unable to bind to EnzIII, a cytoplasmically-located protein of the E. coli phosphotransferase system. Overall, this work should provide considerable information concerning the sugar recognition site, the H+ recognition site, and the structure of the protein within the membrane. Furthermore, the general features which are learned about the molecular mechanism of the H+:lactose carrier may ultimately apply to other cation-substrate cotransporters which are found not only in bacteria, but also in fungi, plant, and animal cells.